SRP orchestrates protein biogenesis beyond initial ER membrane targeting.

The Signal Recognition Particle (SRP) targets nascent proteins to the Sec61 translocon for import into the endoplasmic reticulum (ER). However, its range of substrates, point of engagement during targeting, and hence full biological impact remain unclear. Here, we examined SRP interactions with the nascent proteome of S. cerevisiae during translation and membrane targeting. SRP binds effectively to transmembrane domains (TMDs) as they emerge from the ribosomal tunnel, but only to a minority of cleavable signal peptides. We identify nascent chain features that promote SRP binding, allowing to develop a predictive algorithm. We show SRP performs a role in triaging nascent ER proteins into distinct targeting routes and downstream maturation processes. Furthermore, ribosomes frequently dissociate from the membrane before completing translocation, allowing the chaperone Ssb to assist folding of emerging cytosolic domains. Ribosomes translating multipass membrane proteins are retargeted to the translocon through repeated SRP interactions with internal TMDs, emphasizing collaboration between SRP and chaperones in membrane protein biogenesis.